Compounds for activating TGF-β signaling

ABSTRACT

Compositions and methods for treatment and prevention of disorders and conditions characterized by reduced TGF-β signaling are described.

PRIORITY

The present application claims priority to U.S. Provisional ApplicationSer. No. 60/998,658, filed Oct. 12, 2007, which is herein incorporatedby reference in its entirety.

FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support from the Department ofVeterans Affairs and under contract AG020603 awarded by the NationalInstitutes of Health. The Government has certain rights in thisinvention.

TECHNICAL FIELD

The present subject matter relates to small-molecule compositions andmethods for treatment and prevention of diseases and conditionsassociated with reduced TGF-β signaling, including neurologicaldisorders.

BACKGROUND

TGF-β

Transforming growth factor-β (TGF-β) signaling is implicated in anumerous diseases and conditions, including stroke, heart disease, boneloss, cancer, multiple sclerosis, wound healing, inflammation, andneurodegenerative disorders. TGF-β is a member of a superfamily ofconserved cytokines, growth factors, and morphogens, which play keyfunctions in development and homeostasis.⁹⁻¹¹ The TGF-β subfamilyincludes three isoforms in mammals, TGF-β1, 2 and 3, which promote cellsurvival, induce apoptosis, stimulate cell proliferation, inducedifferentiation, and/or initiate or resolve inflammation, depending onthe particular cell type and environment. Accurate regulation of TGF-βbioactivity and signaling is key to controlling these functions andessential to health and normal aging. The disruption of TGF-β signalingmolecules frequently results in embryonic lethality in mice.^(12,13)

The biological actions of TGF-βs are mediated by a receptor complexconsisting of the TGF-β type 1 (TBR1/ALK5) and type 2 (TBR2)serine/threonine kinase receptor subunits.^(9,10) Receptor activationleads to phosphorylation of Smad proteins, which translocate to thenucleus where they bind to the Smad DNA-binding element (SBE) present inan estimated 400 genes.¹⁴ TGF-βs can also activate other signalingpathways including the p38 MAP kinase pathway and the JNK or NF-kBpathways.¹¹ Despite interaction with other pathways, knockout studies inmice suggest that Smad proteins are the key mediators of many ofTGF-β1's actions in vivo.¹³

TGF-B in the CNS

TGF-β is known to play a role in neurological diseases and condition. Inthe normal central nervous system (CNS), TGF-β1, 2, and 3, and theirreceptors are expressed in neurons, astrocytes, and microglia.^(4,5) Thebest studied isoform, TGF-β1, is expressed in the adult CNSpredominantly in response to CNS injury, and may function as anorganizer of protective and regenerative responses.¹⁵ It is upregulatedin glial cells in response to brain lesioning, transient forebrainischemia, and stroke.⁵ TGF-β2 and TGF-β3 bind to the same receptors asTGF-β1 but have different patterns of activation and expression.^(10,16)Immunoreactivity to TGF-β2 and TGF-β3 is detected in astrocytes andneurons in the normal CNS and is increased in neurodegenerative diseasesor after stroke.^(4,5) Changes in TGF-β expression are reported in ADbrain, cerebrospinal fluid (CSF), and serum.^(3,17-22) TGF-β1immunoreactivity is increased in (or near) amyloid plaques^(19,20) andaround cerebral blood vessels.^(3,18,22)

TGF-β Protection of Neurons

TGF-β1 has been demonstrated to protect neurons against various toxinsand injurious agents in cell culture and in vivo.^(4,5,32) Astroglialoverexpression of TGF-β1 in transgenic mice protected againstneurodegeneration induced with the acute neurotoxin kainic acid orassociated with chronic lack of apolipoprotein E expression.⁷ Boche andcoworkers also demonstrated that TGF-β1 protects neurons fromexcitotoxic death.³⁶

Several mechanisms have been postulated to explain how TGF-β1 protectsneurons. For example, TGF-β1 decreases Bad, a pro-apoptotic member ofthe Bcl-2 family, and contributes to the phosphorylation, and thusinactivation, of Bad by activation of the Erk/MAP kinase pathway.³⁷ Onthe other hand, TGF-β1 increases production of the anti-apoptoticprotein Bcl-2.³⁸ TGF-β1 has also been shown to synergize withneurotrophins and/or be necessary for at least some of the effects of anumber of important growth factors for neurons, including neurotrophins,fibroblast growth factor-2, and glial cell-line derived neurotrophicfactor.^(32,39) In addition, TGF-β1 increases laminin expression⁴⁰ andis necessary for normal laminin protein levels in the brain.⁷ It is alsopossible that TGF-β1 decreases inflammation in the infarction area,attenuating secondary neuronal damage.³⁵

Transgenic Animals

TGF-β1 transgenic mice overexpressing a secreted, constitutively activeform of TGF-β1 in astrocytes at modest levels develop age-relatedcerebrovascular abnormalities including thickening of the capillarybasement membrane and cerebrovascular amyloid deposition,^(22,29)nevertheless, these mice have better cognitive function thannontransgenic controls. Similar microvascular abnormalities are typicalfor AD and consistent with the observation that TGF-β1 mRNA levels inbrains of AD cases correlate positively with vascular amyloiddeposition.²²

TGF-β1 transgenic mice cross-bred with human amyloid precursor (hAPP)transgenic mice, develop synaptic degeneration and amyloid plaques inthe brain parenchyma. Unexpectedly, a prominent reduction in plaqueformation and overall Aβ accumulation was found in hAPP/TGF-β1 doubletransgenic compared with hAPP mice.³ Most of the remaining amyloidaccumulated around cerebral blood vessels.

Increased levels of TGF-β1 reduced the number of plaques in humanamyloid precursor protein (hAPP) mice by 75% and overall Aβ levels by60%, compared to mice with normal levels of TGF-β1. Interestingly,TGF-β1 stimulated microglial cells to degrade synthetic Aβ peptide inculture. Because TGF-β1 also caused an activation of microglia inhAPP/TGF-β1 mice, these data suggest that at least some of the effectsof TGF-β1 involve the activation of microglial phagocytosis.

The need exists for more effective pharmaceutical compounds for treatingand preventing stroke, heart disease, bone loss, cancer, multiplesclerosis, wound healing, inflammation, and neurodegenerative disorders.The present compositions and methods involve small-molecules thatmodulate TGF-β signaling.

REFERENCES

The following reference and any additional references cited herein areincorporated by reference in their entirety.

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SUMMARY

The present composition and methods are for treating and preventingdisorders associated with reduced TGF-β signaling. The following aspectsand embodiments thereof described and illustrated below are meant to beexemplary and illustrative, not limiting in scope.

In one aspect, compound having the following structure is provided:

In some embodiments, R is selected from the group consisting of H, halo,alkyl, and substituted alkyl. In particular embodiments, R is selectedfrom the group consisting of H, F, Cl, CH₃, OCH₃, N(CH₃)₂, and CF₃.

In some embodiments, R₁ is selected from the group consisting of alkyl,allyl, and ether. In particular embodiments, R₁ is a cyclic alkyl. Inparticular embodiments, R₁ is —(CH₂)_(n)—OCH₃, wherein n is 1, 2, or 3.In particular embodiments, R₁ is —(CH₂)_(n)CH₃, wherein n is 1 or 2. Inparticular embodiments, R₁ is —(CH₂)_(n)CH(CH₃)₂, wherein n is 1 or 2.

In some embodiments, R₂ is an aromatic group or a substituted aromaticgroup. In particular embodiments, R₂ is selected from the groupconsisting of thiophene, methylthiophene, and furan. In particularembodiments, R₂ is selected from the group consisting ofp-trifluoromethylphenyl and o-chlorophenyl. In some embodiments, R₂ is abranched alkyl or cyclic alkyl. In some embodiments, R₂ is2-chloropentane or cyclopropyl.

In some embodiments, the tryptamine of the compound is substituted atone or more of the 4, 5, 6, or 7 positions, and wherein the substitutionis independently selected, for each position, from the group consistingof fluoro, methyl, hydroxyl, methoxy, and benzyloxy. In someembodiments, the tryptamine is substituted at one or more of the α or 1positions, and wherein the substitution is independently selected, foreach position, from the group consisting of fluoro, methyl, hydroxyl,methoxy, and benzyloxy.

In particular embodiments, the compound has the structure:

In some embodiments, the compound is a TGF-β agonist.

In another aspect, a compound having the following structure isprovided:

In some embodiments, R₄ is selected from the group consisting ofthiophene, furan, and cyclic alkyl. In some embodiments, R₅ is phenyl orsubstituted phenyl. In some embodiments, R₆ is piperidine orN-substituted piperidine attached via an alkyl group.

In particular embodiments, the compound does not have the structure:

In some embodiments, the compound is a TGF-β agonist.

In another aspect, a compound having the following structure isprovided:

In some embodiments, R₇ is selected from the group consisting of H,halo, phenyl, substituted phenyl, and substituted alkyl. In someembodiments, R₈ is selected from the group consisting of H, thiophenyl,furanyl, phenyl, substituted phenyl, benzyl, substituted benzyl, andsubstituted alkyl. In some embodiments, R₉ is selected from the groupconsisting of H, alkyl, alkyne, and substituted alkyl.

In particular embodiments, the compound has the structure:

In some embodiments, the compound is a TGF-β agonist.

In another aspect, a pharmaceutical composition comprising one or moreof the above compounds is provided. In some embodiments, thepharmaceutical composition is formulated for oral delivery.

In yet a further aspect, a method for increasing TGF-β signalingactivity in a mammalian patient is provided, comprising administeringone or more of the above compounds.

In particular embodiments, the compound is selected from the groupconsisting of:

In some embodiments of the method, the increasing TGF-β signalingactivity occurs in the brain of the mammalian patient.

In some embodiments, the mammalian patient has a disease or conditioncharacterized by reduced TGF-β signaling activity.

In particular embodiments, the disease or condition is selected from thegroup consisting of stroke, heart disease, bone loss, cancer, multiplesclerosis, wound healing, inflammation, and a neurological disorder. Inparticular embodiments, the disease or condition is the disease orcondition is Alzheimer's disease (AD).

In some embodiments, the increasing TGF-β signaling activity enhancesneuroprotection in the brain. In particular embodiments, the compoundreduces the number of amyloid plaques in the brain. In particularembodiments, the compound reduces the accumulation of Aβ in the brain.

In some embodiments, the compound modulates the TGF-β pathway.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1J relate to small molecule compounds with TGF-β signalingactivity and related data. (A) Graph showing relative activity ofcompounds in reporter cells. (B) Chemical structures. (C) Graph showingfold-induction of TGF-β signaling activity in reporter cells. Structuralrendering compounds 3A (D), 11H (G), and 5E (F). (E) Alignedconformations of several agonist compounds. (H) Graph showing neuronsurvival in cells. (I) Graph showing relative gene expression levels ofTGF-β-responsive genes. sulting supernatant was used for luciferaseassays. (J) Graph showing luciferase activity in mouse hippocampiresulting from a TGF-β reporter gene.

FIGS. 2A-2D show a model and results relating to screening a library ofcompounds. (A) Diagram of the TGF-β signaling pathway. (B-D) Graphsshowing activity in transfected cells.

FIGS. 3A-3G show the results of experiments using transgenic reportermice to monitor the activity of TGF-β and an exemplary TGF-β agonist.(A-C) Representative images of mice treated with the indicated drugs atbaseline or 5 h after treatment. (D, E) Graphical representations ofbioluminescence changes (fold-induction) following administration of anexemplary TGF-β analog. FIGS. 3F and 3G show reporter gene expression inhippocampal neurons of reporter mice following administration of vehicle(F) or an exemplary TGF-β agonist (G).

FIGS. 4A-4F show the results of experiments using reporter mice tomonitor brain injury and drug efficacy and treatment of mice with anexemplary TGF-β agonist. (A) Images from sections of mice with differentextent of injury and reporter gene activity. (B-E) Graphs relating toTGF-β activation by an exemplary TGF-β agonist. (F) Graph showing theresults of Aβ immunohistochemistry.

FIG. 5A illustrates regions of the 3A compound. FIG. 5B shows a 3-Dspace filling model of 3A.

FIG. 6A illustrate regions of the 11H compound. FIG. 6B shows a 3-Dspace filling model of 11H.

FIGS. 7A and 7B illustrate synthesis schemes for compounds 3A and 11H,respectively.

FIG. 8A is a graph showing the results of Morris water maze testing.

FIG. 8B is a graph relating to HDAC.

DETAILED DESCRIPTION I. Introduction

In one aspect, compositions are provided for treating a mammalianpatient at risk for or diagnosed with a disease or conditioncharacterized by reduced TGF-β signaling. The compositions arebioactive, small-molecule, TGF-β agonist compounds that cross the bloodbrain barrier (BBB). Reduction in TGF-β signaling is associated withsuch diseases and conditions as stroke, heart disease, bone loss,cancer, multiple sclerosis, wound healing, inflammation, andneurodegenerative disorders. TGF-β agonists are known to reduce thenumber of amyloid plaques and overall accumulation of Aβ in the ADbrain.

In another aspect, methods are provided for treating or preventing theprogression of a disease or condition characterized by a reduction inTGF-β signaling. On category of such diseases or conditions areneurodegenerative disorders. One example is Alzheimer's disease (AD), oranother disease characterized by the deposition of amyloid plaques, andoverall accumulation of Aβ in the brain. Examples of such diseases aredescribed, infra.

Experiments performed in support of the present compositions and methodsare described, below.

II. TGF-β Reporter Gene and Cells for Screening Compounds

An in vitro screening method was developed to identify small-moleculechemical compounds with TGF-β1-like bioactivity. The method utilized afusion gene consisting of the luciferase or secreted alkalinephosphatase (SEAP) reporter gene under control of the TGF-β responsiveSmad-binding element (SBE) minimal promoter sequence.⁵² To screen forcompounds that mimic TGF-β bioactivity, reporter cell lines wereprepared by stably-integrating SBE-SEAP into C6 astrocytoma cells, aprimary astrocyte line derived from tgfb1^(−/−) mice, a mouse embryonicfibroblast cell line derived from tgfb1^(−/−) mice (MFB-F11), andNG108-15 neuroblastoma cells (Table 1). Such cell lines remained stablefor at least 20 passages.⁵³ Use of a tgfb1^(−/−) genetic backgroundavoids interference by the exogenous tgfb1 gene.

TABLE 1 Cell lines for screening of compounds with TGF-β signalingactivity Reporter Colonies/ Lines Average fold Cell line Origin genelines tested selected induction** C6 Rat astrocytoma SBE-SEAP* 34 6 7(line 29) MFB Mouse fibroblast, Tgfb1^(−/−) SBE-SEAP 49 7 >500 (lineF11) MAB39 Mouse primary SBE-SEAP 54 2 3 (line 66) astrocytes,Tgfb1^(−/−) NG108-15 Mouse neuroblastoma SBE-SEAP 46 5 18 (line H18)*using 1 ng/ml TGF-β1, # SBE, Smad binding element; SEAP, secretedalkaline phosphatase **best cell line *

III. Initial Compound Screening

A library with 5,000 chemically diverse small molecules (average Mr 200Da) was obtained from Comgenex LLC (South San Francisco, Calif.). Totest compounds, or to assay individual stably transfected cell lines forresponsiveness to TGF-β1, 4×10⁴ cells were seeded in 10% FBS/DMEM into96-well plates and incubated overnight, washed twice, and exposed totest compounds (at 4 or 20 μM) or recombinant TGF-β1 (at 0 to 1,000pg/ml) in serum free medium (DMEM). Conditioned medium was assayed forSEAP activity 24 and 48 hours later.

All compounds were tested twice, in duplicate, using the MFB-F11 cellline. 80 compounds were selected for further study based on induction ofthe reporter gene in any of the replicates. These compounds were testedagain in MFB-F11 or C6-H4 cells, either alone or in the presence of 100pg/ml of TGF-β1, to test for synergistic activities. Several compoundsshowed activities of up to an equivalent of 100 pg/ml TGF-β1 at 20 μM inthe TGF-β1 deficient MFB-F11 cells. Some compounds showed dose-dependentactivities. The most promising compounds (i.e., the three most active“hits”) were 3A, 11H, and 5E, having the propose IUPAC namesN-(2-(1H-indol-3-yl)ethyl)-N-benzyl-2-(N-(2-methoxyethyl)-2-(thiophen-2-yl)acetamido)acetamide,(E)-3-cyclohexyl-1-(2-methyl-3-phenylallyl)-1-(piperidin-4-ylmethyl)urea,and ethyl2-(3-(4-(4-(2-methoxyphenyl)piperazin-1-yl)-3-(1-phenylethylcarbamoyl)phenyl)ureido)acetate,respectively, and the following structures:

Data relating to these three lead compounds are shown in FIGS. 1A-1J.FIG. 1A is a graph showing the relative activity of compounds 3A, 11H,and 5E following independent addition to MFB-F11 reporter cells at 20 μM(3A, 5E) or 10 μM (11H). SEAP activity was compared to a standard curvegenerated with cells exposed to different concentrations of recombinantTGF-β1. The chemical structures of 3A, 11H, and 5E are, shown in FIG.1B. FIG. 1C is a graph showing fold-induction of TGF-β signalingactivity in MFB-F11 reporter cells by compound 3A and 7 analogs (scale 0to 25 fold) and compound 11H (circles, stippled line; scale 0 to 5).FIGS. 1D, 1F, and 1G show structural renderings of compounds 3A, 11H,and 5E, respectively.

FIG. 1H is a graph showing the relative amount of surviving 6-7 DIVhippocampal neurons of E16 mice following exposure to culture medium(CM) alone, 13A04 (3 μM), or CM containing Aβ at 2 or 5 μM with TGF-β1(10 ng/ml) or 13A04 (3 μM). After 24 hours, cultures were fixed andsurviving cells were counted. Data represent mean±SEM, n=3-7 fieldscounted per plate. All conditions were compared to Aβ using theMann-Whitney test (* p<0.05, ** p<0.01). FIG. 1I is a graph showing therelative gene expression levels of the indicated TGF-β-responsive genesinduced in MFB-F11 fibroblasts stimulated with TGF-β1 (yellow) or 13A04(blue) for 24 h. mRNA was extracted, labeled and hybridized to geneexpression arrays tailored for the TGF-β pathway. FIG. 1J showsactivation of a TGF-β reporter gene in the hippocampi of mice injectedwith 11H. These data demonstrate that compounds 3A, 11H, and 5E areTGF-β-agonists.

Compounds 3A and 5E were not found in a searchable drug database.Compound 11H is distributed by Comgenex (the provider of the library).

IV. Second Compound Screening

Compounds 3A, 5E, and 11H from the initial screening were analyzed usingthe GASP pharmacophore modeling program, which is part of the Sybylmolecular modeling software (Tripos, Inc). GASP allows mapping theimportant functional elements into three-dimensional space without priorknowledge of geometric constraints or receptor structure. The buildingof a three-dimensional pharmacophore model allows the rational design ofsmall-molecule inhibitors with modified flexibility constraints and/orhydrophobic and electrostatic properties.

A. Compounds Based on the 3A Core

The energy-minimized structure of compound 3A revealed a uniqueconformation as shown in FIG. 1D. The three most active compounds (i.e.,13A04, 22B02, and 3A) assume similar conformations when docked (FIG.1E). Several analogs of 3A were able to activate the TGF-β reporter inMFB-F11 cells, some of them with higher potency than the originalcompound (FIG. 1C and Tables 2A and 2B). Induction of the TGF-β reportergene ranged from 20.9 fold to no induction.

Tables 2A and 2B shows the structures and biological properties,respectively, of the additional compounds/hits related to compound 3A(highlighted in grey). 3A-based compounds are based on the followingcore structure:

TABLE 2A Structure activity of lead compounds related to 3A.

Com- pound R R₁ R₂ 13A04 —H —CH₂CH(CH₃)₂

22B02 —CH₃ (p) —(CH₂)₂—OCH₃

3A —H —(CH₂)₂—OCH₃

22E02 —F (p) —(CH₂)₃—OCH₃

21B08 —CF₃ (p) —(CH₂)₃—OCH₃

13F03 —F (p) —(CH₂)₂—OCH₃

22D03 —F (p) —(CH₂)₃—OCH₃

21F11 —CF₃ (p) —CH(CH₃)₂

23C08 —N(CH₃)₂ (p) —CH₂CH(CH₃)₂

37B02 —OCH₃ (o) —(CH₂)₂—OCH₃

23D05 —N(CH₃)₂ (p) —CH(CH₃)₂

23B08 —N(CH₃)₂ (p) —CH(CH₃)₂

23A08 —N(CH₃)₂ (p) —CH(CH₃)₂

37C02 -3,4-Cl —CH(CH₃)₂

21A11 —CF₃ (p)

44A04 —H

21C09 —CF₃ (p) —(CH₂)₂—OCH₃

22B05 —F (p) —(CH₂)₂—OCH₃

13G03 —OCH₃ (o)

21A08 —CF₃ (p) —(CH₂)₃—OCH₃

24D04 -3,4-OCH₃ —(CH₂)₂—OCH₃

Relative TGF-β activity in the cell reporter assay at four differentconcentrations and calculated log P, tPSA (topological polar surfacearea; a measure of passive molecular transport through membranes,⁵⁹ andlog BB (a measure of blood-brain barrier penetration⁶⁰) are shown inTable 2B. A tPSA of <60 and C log P of from about 1.5 to about 2.5 arepreferred.

TABLE 2B Structure activity of lead compounds related to 3A. RelativeTGF-β activity* 100 μM 30 μM 10 μM 3 μM CLogP tPSA Log BB 13A04 20.917.6 6.3 2.5 4.85 76.9 −0.26 22B02 14.1 5.7 2.9 1.4 4.34 76.9 −0.34 3A11.4 5.2 2.9 1.7 4.46 76.9 −0.32 22E02 9.1 8.6 4.7 1.4 6.09 76.9 −0.0721B08 9.1 7.0 4.8 1.6 5.59 76.9 −0.15 13F03 9.1 3.8 1.9 1.3 4.77 76.9−0.27 22D03 8.8 8.3 3.7 1.4 5.86 76.9 −0.11 21F11 6.5 2.7 1.6 0.8 5.40110.1 −0.67 23C08 5.4 3.6 3.1 1.3 6.06 65.5 0.09 37B02 5.2 4.7 1.9 1.14.82 91.0 −0.48 23D05 4.7 1.7 1.1 0.8 5.15 101.9 −0.59 23B08 4.5 3.1 1.81.1 5.25 65.5 −0.03 23A08 3.4 2.8 2.3 1.2 5.47 93.7 −0.42 37C02 3.1 1.41.0 0.7 6.93 62.8 0.26 21A11 2.9 2.3 2.3 1.3 5.67 76.9 −0.14 44A04 2.82.7 2.3 1.2 4.58 62.8 −0.09 21C09 2.8 1.9 1.7 1.0 6.46 76.9 −0.02 22B052.8 1.7 1.2 1.1 5.32 124.2 −0.89 13G03 2.7 2.6 2.5 1.5 4.27 76.9 −0.3521A08 2.7 1.9 1.5 1.1 6.59 76.9 0.00 24D04 2.7 1.5 1.1 1.2 4.47 105.1−0.74 *Fold change compared with vehicle

Docking of several of the most active compounds, as exemplified, in FIG.1E, suggested that the 3A-related compounds included four regions thatcan be modified to tailor the compound a particular application. Forexample, structural features that enhance activity and oralbioavailability can be combined in the same compound. The four regionsare shown in FIG. 5A. A three-dimensional, space-filling model ofalignments is shown in FIG. 5B.

The tryptamine in Region A remained unchanged within the tested seriesof compounds; however, a 5-fluoro-, 5-chloro-, 5-methyl-, 5-hydroxy-,5-methoxy, 5-benzyloxy, or similar substitutions at one or more of the4, 6, or 7 positions of the ring structure are contemplated. α-methyland 1-methyl tryptamine substitutions are also contemplated. Otherstructural modifications that are expected to produce compounds withsuitable activity and pharmacokinetic properties (i.e., based on theresults shown in Tables 2A and 2B) are shown in Table 3, where R, R₁,and R₂ are as indicated in the above 3A core structure and in FIG. 5A.

TABLE 3 Exemplary R groups for attachment to the 3A core. R R₁ R₂ —H—CH₂CH(CH₃)₂

—CF₃ —CH₂CH(CH₃)₂

—H —CH₂N(CH₃)₂

—CF₃ —(CH₂)₃—OCH₃

—F —(CH₂)₃—OCH₃

—N(CH₃)₂ —(CH₂)₂—OCH₃

—H —(CH₂)₂—OCH₃

From the screening data it appears that region B should be aromatic,with limited substitution, which may include basic groups such asdimethyl amine. Activity is lost, when R has a bulky substitution and R₂is also bulky; therefore, small heterocyclic rings or minimallysubstituted aromatic rings are proposed for region B. Region C and R₁should be small and lipophilic or a straight chain alcohol. The additionof a ring system connecting regions A and B is also contemplated, andexpected to reduce flexibility, constrain the three-dimensionalconfiguration, and possibly increase activity. In one example, the A andB regions are linked using a backbone harboring a β-carboline to whichadditional R groups can be added.

An exemplary process of making the 3A compound is shown as Scheme I inFIG. 7A. Intermediate compounds 3-5 also expected to possess suitableactivity and pharmacokinetic properties.

B. Compounds Based on the 11H Core

FIG. 6A shows three regions of the 11H-based compounds that can bemodified to tailor the compound a particular application (based onalignments and structure-activity analyses, as discussed above). Athree-dimensional, space-filling model is shown in FIG. 6B. Based on thecore structure shown below, the R groups exemplified in Table 4 areexpected to produce active compounds.

TABLE 4 Compounds based on the 11H core.

Compound R₄ R₅ R₆ CGX-0540523

CGX-0540547

CGX-0540672

CGX-0541586

An exemplary scheme for synthesizing a second series of compoundsrelated to compound/hit 11H is shown as Scheme II in FIG. 7B.Intermediates in the synthesis are expected to have activity similar tothat of 11H.

C. Compounds Based on the 5E Core

Several 5E-related compounds have also been synthesized for testing inthe described assays. Table 5 identifies R groups expected to produce anactive compound, based on the following core structure:

TABLE 5 Compounds based on the 5E core

Name R₇ R₈ R₉ CGX-0730795 —CH(CH₃)₂

CGX-0731125 —(CH₂)₅

—N(CH₃)₂ CGX-00731278

CGX-0731359

CGX-0731462

CGX-0733464 —CH₂CH(CH₃)₂

CGX-0733818 —(CH₂)₅

CGX-0733942

CGX-0740410 —CH₂CH₂CH(CH₂CH₃)₂

CGX-0740492

CGX-0740613 —CHCH₃(CH₂CH₃)

CGX-0740807

CGX-0740885 —CH₂CH₂CH₃

CGX-0741048

CGX-0730795 —CH(CH₃)₂

CGX-0731125 —(CH₂)₅

—N(CH₃)₂ CGX-00731278

CGX-0731359

CGX-0731462

CGX-0733464 —CH₂CH(CH₃)₂

CGX-0733818 —(CH₂)₅

CGX-0741060

—CH₂CO₂CH₂CH₃

CGX-0741107

CGX-0741157

CGX-0741186

CGX-0741210 —(CH₂)₂OCH₃

CGX-0741328

—CH₂CO₂CH₂CH₃

CGX-0746144 —(CH₂)₂CHCH₃(CH₂)₂—

CGX-0730795 —CH(CH₃)₂

CGX-0731125 —(CH₂)₅

—N(CH₃)₂ CGX-00731278

CGX-0731359

CGX-0731462

CGX-0733464 —CH₂CH(CH₃)₂

CGX-0733818 —(CH₂)₅

CGX-0746262 —(CH₂)₃OCH₃

CGX-0746351 —(CH₂CH₃)₂

CGX-0746781

CGX-0746908

CGX-0747015 —(CH₂)₂OCH₃

CGX-0747197

CGX-0747389 —(CH₂)₃OCH₃

CGX-0730795 —CH(CH₃)₂

CGX-0731125 —(CH₂)₅

—N(CH₃)₂ CGX-00731278

CGX-0731359

CGX-0731462

CGX-0733464 —CH₂CH(CH₃)₂

CGX-0733818 —(CH₂)₅

CGX-0752302 —CHCH₃CH₂CH₃ —C(CH₃)₃

CGX-0752382 —(CH₂)₅

CGX-3006045

—N(CH₃)₂

Compounds identified in the above screens, and subsequent screens, canbe tested any of the in vitro and in vivo assays described herein andknown in the art, including the reporter cell screening assay,neurological protection assay, bioavailabilty assay, toxicity assay,ADMET (i.e., absorption, distribution, metabolism, and excretion)assays, blood-brain barrier assay, in vivo/transgenic animal assays, andbehavioral assays, described above and in Examples.

The present compositions and methods include salts of active compoundsand formulations containing active compounds.

V. Effects of TGF-β Analogs In Vitro

Some of the small-molecule TGF-β agonist compounds were tested forneuroprotection activity and toxicity in vitro (cell culture). Inaddition, the mechanism of action of the agonists was explored usingcells with different genetic backgrounds with respect to protein of theTGF-β1 signaling pathway.

A. Neuroprotection Activity

FIG. 1H shows representative data from several experiments in which Aβoligomers are used at different concentrations to induce 50-70% celldeath in cultures of primary hippocampal neurons from E16 mouse embryos.TGF-β1 and a small molecule TGF-β agonist (13A04) consistently protectedneurons and increased survival. Moreover, the small-molecule TGF-βagonist was not toxic at 3 μM and 10 μM (not shown). These resultsdemonstrated that small-molecule TGF-β signaling agonists, such as13A04, were active in primary neurons and protected them against Aβtoxicity.

B. Toxicity

A microarray assay was used to identify changes in gene expressioncaused by stimulating tgfb1^(−/−) fibroblasts with TGF-β1 or 13A04. mRNAwas harvested 1, 5, and 24 hours following stimulation of tgfb1^(−/−)fibroblasts with TGF-β1 (or control) and analyzed for differentialexpression of 128 genes related to TGF-β signaling, using a commerciallyavailable filter array (Superarray Bioscience Corp.; FIG. 1I). While theexpression of many genes was induced by TGF-β1 and 13A04 treatment(e.g., Fos, Jun, Myc, Smad1, Smad2, Smad3, Interleukin-6, etc.) theexpression of other genes was differentially by TGF-β1 and 13A04 (e.g.,Bmper, Col1a1, Fkbp1b, Serpine1 (aka PAI-1), Plat, etc.). Several genesin the latter group have important roles in vascular remodeling. Thesedata suggest that 13A04 (and similar analogs) are agonists of the TGF-βsignaling pathway, mimicking the actions of TGF-β1. However, there maybe additional clinical advantages, e.g., involving vascular remodeling,of the small-molecule agonists.

C. Mechanism of Action/Targets

To understand how the TGF-β signaling agonists function and identifymolecular genetic targets in cells, fibroblasts lacking differentelements of the TGF-β signaling pathway, including Smad2, Smad3, andSmad4, were used in combination with plasmids encoding differentinhibitors of the TGF-β signaling pathway to study TGF-β agonists indifferent backgrounds. For reference, the canonical TGF-β signalingpathway is illustrated in FIG. 2A. The biological actions of TGF-β1 aremediated through interactions with the type 2 TGF beta receptor (TBR2) 2and the type I TGF beta receptor (TBRI) 3. Betaglycan 4 may participatein the binding of TGF-β1. Receptor activation leads to thephosphorylation of Smad proteins, such as Smad 2/3 5 and Smad 4 6, whichtranslocate to the nucleus 7 to bind to the Smad, DNA-binding element(SBE) 8, present in numerous genes, in combination with transcriptionfactors (TF) 9. Smad 7 10 is inhibitory with respect to Smad 2/3 5phosphorylation. Alternative signaling involves p38 11 and the MAPkinase pathway. TAB1 12 activates TAK1 13, which activates p38 11.

The signaling of recombinant TGF-β1 is blocked or strongly inhibited bythe absence of Smad4 or the expression of a dominant negative (dn) TGF-βreceptor, dominant negative Smad 3, or Smad 7 (FIGS. 2B-2D). Signalingby the 3A agonist was not affected (or was even stronger) in the absenceof Smad4 (FIG. 2D).

Because Smad2 and Smad3 can substitute for each other, results withSmad2 or Smad3 knock-out fibroblasts were more difficult to interpret.Nonetheless, the synergistic effect of TGF-β1 and 11H was blocked in theabsence of Smad2 (FIG. 2B) or Smad3 (not shown). In these assays,signaling is reported via SBE elements, which are specific elements thatbind Smad proteins. Thus, the 11H compound must signal through thiselement. Without being limited to theory, it is believed that the 3Acompound functions as a competitive agonist of Smad binding to the SBE,representing a novel target and mode of action.

VI. Animal Studies

Transgenic mice harboring the SBE-luc reporter gene were generated andused to test the present compositions in vivo. These “reporter mice”mice express SBE-luc (i.e., “the transgene”) in all cells of thebody.^(1,54) Intraperitoneal injection of luciferin into SBE-luc miceproduces bioluminescence generated by luciferase and photons penetratingthe skull can be imaged using sensitive camera systems.^(1,54)

A. Monitoring TGF-β Pathway Activation in Animals

Tissue analysis of transgene expression at basal level in 2-month-oldmice of lines T9-55 and T9-7 showed the highest levels of luciferaseactivity in the brain. Further dissection of the brain into differentregions (not shown) demonstrated the highest levels of reporter activityin the hippocampus and cortex.

Primary astrocytes and primary neurons isolated from the brains of theSBE-luc mice showed increased expression of luciferase in response torecombinant TGF-β1.¹ Luciferase activation corresponded with nucleartranslocation of activated (i.e., phosphorylated) Smad2, which is atranscription factor and downstream signal transducer of the TGF-βsignaling pathway (not shown).

These experiments demonstrated that the reporter mice show the expecteddistribution of TGF-β1 in the body and that the mice responded asexpected to exogenous TGF-β1. The transgene can be used to monitoractivation of the TGF-β signaling pathway in vivo using living mice.

B. Bioactivity and Availability

To determine whether 3A and its analogs are bioactive in vivo and ableto penetrate the blood brain barrier (BBB), TGF-β1, 3A, 22B02, or 13A04were injected into reporter mice i.p. or s.q. The animals were thensacrificed at the indicated times following injection. Bioactivity wasmeasured in living mice using bioluminescence as shown in FIGS. 3A-3C.FIG. 3D summarizes the data, and shows that activation by 13A04 was in adose-dependent manner. There was no obvious activation of the reporterin other organs.

Bioactivity data were confirmed by biochemical measurements of tissueluciferase in hippocampal homogenates (FIG. 3E). The results showed thatperipheral administration of recombinant TGF-β1 and 3A-like compoundsactivated TGF-β signaling in vivo, indicating that the compounds passthe BBB.

C. TGF-β Signaling at the Cellular Level

To study TGF-β signaling at the cellular level, additional reporter micewere generated, which express a fusion protein consisting of luciferase,red fluorescent protein (RFP), and thymidine kinase (TK) under controlof the SBE promoter (i.e., SBE-lucRT mice). Using RFPimmunohistochemistry, it was shown that a TGF-β agonist (compound 13A04)activates the reporter gene and TGF-β signaling in hippocampal neurons(FIGS. 3F and 3G). These data are consistent with previous data showingexpression of phosphorylated Smad2 in the same neurons of unmanipulatedor injured wild-type mice. No activation of TGF-β signaling was observedin the vasculature. These results demonstrated that the TGF-β agonistactivated TGF-β signaling in neurons in vivo.

D. Activation of TGF-β Signaling in Response to Excitotoxic Injury

SBE-luc mice were further used for non-invasive monitoring of reporteractivation in response to excitotoxic injury. The mice were lesionedwith kainate in a model for excitotoxic injury (FIG. 4A-4D).⁵⁴Excitotoxicity causes an excessive activation of glutamate receptors andsubsequent Ca⁺⁺ influx in neurons. The resulting neuronal injury leadsto an inflammatory response and oxidative stress that amplifies neuronaldegeneration and death.⁷⁵ Excitotoxicity has been postulated to beresponsible, not only for neuronal loss associated with seizures,traumatic and ischemic brain injury, and hypoxia,⁷⁶ but also forneuronal damage in Alzheimer's disease and other neurodegenerativedisorders.⁵⁵

Relative luciferase reporter activity was measured in kainate lesionedSBE-luc mice as bioluminescence over the skull and compared withpostmortem neuropathological parameters). The relative number of photonsemitted from the brain in lesioned animals correlated with biochemicalmeasurements of luciferase activity in postmortem hippocampal samples(not shown).

Neurodegeneration and microglial activation were most prominent inhippocampus and cortex although the extent of damage varied among mice.This variability is typical for kainate lesions and was advantages incorrelating reporter gene activation with different degrees of damage.Bioluminescence corresponded to silver staining, which is a sensitivemethod for detecting degenerating cells as well as the relative numberof pyramidal neurons estimated in cresyl violet-stained sections (FIG.4A). Similar correlations were found with NeuN, MAP-2, synaptophysin,and calbindin immunostaining, which were used to assesssynapto-dendritic and overall neuronal integrity.

Glial cells were sensitive to neuronal dysfunction and damage.Microglial activation measured as a function of macrosialin/CD68expression showed highly significant positive correlations withbioluminescence (FIG. 4B). These data demonstrated that reporter geneactivity correlated with neurodegeneration and microgliosis in kainateinjured SBE-luc mice, further validating the animal model.

SBE-luc mice can also be used to monitor activation of TGF-β signalingin response to glial activation after lipopolysaccharide (LPS)administration. While LPS induces rapid and long-lasting activation ofTGF-β signaling in the brain, treatment with anti-inflammatory drugssuch as dexamethasone or flurbiprofen strongly reduced signaling (FIG.4E) and microglial activation (data not shown). Bioluminescence imagingin living mice demonstrated the efficacy of these compounds in reducingsignaling, validating the animal model.

In a further experiment, a group of 5-month-old APP-T41 mice weretreated with vehicle or 13A04 (i.p.) for 10 days. At this age, the miceare starting to deposit Aβ which can be quantified in brain sectionsusing Aβ immunostaining. The TGF-β agonist compound was well toleratedand no side effects were observed. Although the result were notstatistically significant, a trend towards reduced Aβ deposition in13A04-treated mice was observed, when compared with controls (FIG. 4F).

E. Activation of TGF-β Signaling in the Hippocampus

In another experiment performed to assess the ability of the presentcompounds to activate TGF-β signaling in animals, SBE-luc mice weregiven either PBS or the 11H compound via i.p. injections as described inExample 10. Following drug or PBS injection, mice were anesthetized withchloral hydrate and perfused with saline. Brains were dissected andsnap-frozen. Hippocampal tissues were assayed for luciferase activity.

Hippocampi from mice injected with the 11H compound demonstrated twicethe levels of activation of the TGF-β reporter gene compared to thoseinjected with PBS (FIG. 1J), demonstrating the activity of the presentcompounds in increasing TGF-β signaling, in vivo.

VII. Summary of Results

Experiments performed in support of the present compositions and methodsshow that TGF-β1 is neuroprotective and can reduce the accumulation ofAβ in an animal model. Small-molecule agonists of TGF-β1 modulated TGF-βsignaling and activated a reporter gene in the brains of transgenicmice. Together, these data suggest that agonists of the TGF-β signalingpathway can be used to reduce or prevent amyloid plaques and Aβaccumulation in the CNS, thereby treating or preventing Alzheimer'sdisease (AD) and associated diseases. While the present compositions andmethods relate primarily to TGF-β1, one skilled in the art willrecognize that TGF-β2 and β3 can be modulated in a similar manner.

While the present compositions and methods are described mainly fortreatment and prevention of AD, other neurodegenerative diseases arecharacterized by amyloid plaques and/or accumulation of Aβ in the braincan be treated or prevented in a similar manner. Such diseases/disordersinclude localized amyloidosis while as well as systemic amyloidosis.Amyloidosis can appear without other pathology or can accompany plasmacell dyscrasia or multiple myeloma. Amyloidosis is also associated withchronic infection or chronic inflammation. Familial forms of amyloidosisinclude, familial Mediterranean fever (FMF), familial British dementia(FBD), and familial amyloid polyneuropathy (FAP). Another form ofamyloidosis is found in long-term hemodialysis patients.Creuzfeldt-Jakob disease, motor neuron diseases, polyglutamine disorders(including Huntington's disease), progressive frontotemporal dementia(FTD), Lewy Body dementia (LB), progressive supranuclear Palsy (PSP),Pick's disease, and Parkinson's disease can also be associated withamyloid plaques and/or accumulation of Aβ in the brain.

Chemical compounds with TGF-β agonist activity have previously beendescribed (e.g., exemplified by A-161906 from Abbott Laboratories). Suchcompounds turned out to be hystone deacetylase (HDAC) inhibitors. Acommercially available assay was used to measure HDAC inhibitoryactivity of the present small-molecule compounds. The present TGF-βagonists did not inhibit HDAC activity in HeLa nuclear extracts, whilethe known inhibitor, trichostatin A, reduced activity by >90% (FIG. 8B).Therefore the present compounds do not appear to be significantinhibitors of HDAC 1, HDAC 2, or SIRT 1 (the main HDACs tested in theassay). In contrast, the TGF-β agonist/HDAC inhibitor, A-161906(Abbott), inhibited HDAC 1 and 2 with an IC50 of 9 nM.

VIII. Methods or Treatment

The present compositions are useful in the preparation of a medicamentfor treating or preventing diseases and conditions associated withreduced TGF-β signaling, including neurological disorders. In aparticular example, the compositions are useful in treating orpreventing AD or other diseases characterized by neurodegeneration. Insome embodiments, the composition is provided in a pharmaceuticalexcipient suitable for oral delivery. In some embodiments, thecomposition is provided in a pharmaceutical excipient suitable for i.p.,i.v., or i.m. delivery.

Tablets and capsules containing a compound for modulating the TGF-βpathway may be prepared by combining a compound with additives such aspharmaceutically acceptable carriers (e.g., lactose, corn starch,microcrystalline cellulose, sucrose), binders (e.g., alpha-form starch,methylcellulose, carboxymethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinylpyrrolidone), disintegratingagents (e.g., carboxymethylcellulose calcium, starch, low substitutedhydroxy-propylcellulose), surfactants (e.g., TWEEN® 80, polyoxyethylenepolyoxypropylene copolymer), antioxidants (e.g., L cysteine, sodiumsulfite, sodium ascorbate), lubricants (e.g., magnesium stearate, talc),or the like.

The present compounds can also be mixed with a solid, pulverulent orother carrier, for example lactose, saccharose, sorbitol, mannitol,starch, such as potato starch, corn starch, millopectine, cellulosederivative or gelatine, and may also include lubricants, such asmagnesium or calcium stearate, or polyethylene glycol waxes compressedto the formation of tablets. By using several layers of the carrier ordiluent, tablets operating with slow release can be prepared.

Liquid preparations for oral administration can be made in the form ofelixirs, syrups or suspensions containing, e.g., ethanol, water,glycerol, propylene, glycol and possibly other additives of aconventional nature.

Other suitable formulations include a protective coating that protectsthe compound in the stomach and intestines until absorbed by theintestinal mucosa. Protective dosage forms for proteins are known in theart, and include enteric coatings and/or mucoadhesive polymer coatings.Exemplary mucoadhesive polymer formulations include ethyl cellulose,hydroxypropylmethylcellulose, Eudragit®, carboxyvinyl polymer, carbomer,and the like.

While the present compositions and methods have been described usingparticular experimental data and examples, further embodiments and usesof the compositions and methods will be apparent to the skilled artisanin view of the disclosure.

EXAMPLES

The following Examples are provided to illustrate the presentcompositions and methods and are in no way intended to be limiting.

Example 1 Iterative Reporter Cell Screen Assays

Compounds are tested for activation of the TGF-β signaling pathway usingtwo different cell lines produced by stably transfecting cells with theSBE-SEAP reporter gene. These cells lines are TGF-β1 knockoutfibroblasts MFB-F11 and mouse NG108-15 neuroblastoma cells. MFB-F11cells do not produce TGF-β1 and are extremely sensitive to activators ofthe pathway.⁵³ Mouse NG108-15 neuroblastoma cells have been usedextensively in the field (>1,100 references in Medline) because they canbe differentiated into cells with neuron-like properties. Such cells areideal for assaying TGF-β1 pathway activity in vitro.

Example 2 In Vitro ADMET Assays

Absorption, distribution, metabolism, and excretion (i.e., ADMET)assays, including metabolic stability assays using human livermicrosomes, permeability assays modeling intestinal and blood brainbarrier absorption, and drug interaction assays that evaluate cytochromeP450 inhibition, are known in the art^(62,63) and can be used toevaluate any of the above compounds and those apparent in view of thesecompounds. Compounds that show favorable characteristics in these invitro assays can be further evaluated for oral bioavailability andtoxicity (maximum tolerated dose) in vivo.

Example 3 Neuroprotection Assays

Primary mixed neuronal cultures are exposed to Aβ oligomers and treatedwith compounds (0 to 10 μM concentration) and cell survival is measuredusing lactate dehydrogenase (LDH) release or by counting4,6-diamidino-2-phenylindole (DAPI)-stained cells. Aβ oligomers may beproduced according to a protocol described by LaDu and coworkers.⁶⁹ Suchpreparations contain mostly oliogomeric Aβ, as demonstrated using atomicforce microscopy, and it is toxic to neurons. Neuritic dystrophy in 21DIV neurons can also be measured by quantifying the tortuosity ofneurites as described by Ferreira et al.⁷⁰ and modified by others. FIG.1C shows the effect of Aβ on these cells. Further incubation of thesecells with the present compounds provides a sensitive assay forneuroprotection.

Example 4 Bioavailability in Animals

Male Sprague-Dawley Rats

Oral bioavailability and BBB passage of test compounds can be measuredusing, e.g., male Sprague-Dawley rats. Animal brains are collected fromrats at preselected times following administration of a test compound orcontrol and a bioanalytical method, e.g., LC/MS-MS, is used to determinebioavailability by comparing the plasma level curves and the brainconcentration of the parent drugs after, e.g., i.v. or oraladministration. All data from these studies will be analyzed usingWinNonlin (SCI Software, NC) or a similar software program to determineappropriate pharmacokinetic parameters such as terminal eliminationhalf-life, area under the curve (AUC), maximum concentration in bloodand/or plasma after oral administration (C_(max)), and otherpharmacokinetic parameters as appropriate. These experiments will beused to determine the % bioavailability of compounds in plasma and brainand show which compounds are orally active and enter the blood.

SBE-Luciferase Transgenic Mice

Two-month old SBE-luciferase transgenic mice receive differentconcentrations of a test compound either s.q., i.p., or orally bygavage. Compound doses may be estimated from in vitro potency, and aretypically in the range of from about 1 to about 50 mg/kg body weight.Following administration of the test or control compounds, mice areinjected with luciferin i.p. At, e.g., 2, 8, and 16 hours followinginjection of luciferin, the mice are imaged to detect bioluminescence asdescribed above. Brains may be harvested 16 hours following testcompound injection, and may be divided sagittally. For example, onehemi-brain can be homogenized in luciferase assay buffer for measurementof reporter gene activation, while the other hemi-brain can be frozen at−70° C. for future study, including sectioning into individual brainregions.

Using this assay, compounds can be tested for the optimal route ofinjection, half-life, bioactivity, toxicity, efficiency of crossing theblood brain barrier (BBB), accumulation in particular regions of thebrain, etc.

Example 5 Toxicity in Animals

Toxicity of test compounds can be determined in male and femaleSprague-Dawley rats. Dose levels are estimated based onstructure-activity analysis, comparison to toxicity of similar drugs,data obtained in vitro, and data obtained in other in vivo studies. Arange of doses covering at least one log are typically employed. Threerats/sex/dose group are administered, e.g., a single oral or i.p. doseof test compound on Day 1 in an appropriate vehicle (e.g., water,methylcellulose, corn oil). In some examples, three dose levels areevaluated for each test compound, along with an appropriate control(e.g., vehicle). The rats are euthanized and necropsied on Day 5.Endpoints include daily clinical observations, body weights, clinicalpathology and gross pathology at necropsy. These studies show whethercompounds have unusual toxicity in a particular organ and help establisha maximal therapeutic dose.

Example 6 Excitotoxic Injury Model

Mice are injured with kainic acid and treated with different testcompounds at two concentrations each to determine if they can reduceneurodegeneration and/or microglial activation. Wildtype mice on theFVB/N genetic background (8-weeks-old) will be injured with kainic acid(Tocris, Ellisville, Mo.) dissolved in PBS and injected subcutaneously(s.q., 20 mg/kg).⁵⁴ Seizure activity is scored from 0 to 5, with 0corresponding to no behavioral changes and 5 corresponding to constantrearing and falling.⁷⁷ Only kainate-injected mice reaching at leaststage 3 are used for the studies. On day five following injury, mice areanesthetized, transcardially perfused with 0.9% saline, and brainsharvested and dissected. One hemi-brain may be fixed for 24 hours in 4%paraformaldehyde and cryoprotected in 30% sucrose. Serial coronalsections (40 μm) can be cut with a freezing microtome (Leica, Allendale,N.J.) and stored in cryoprotective medium. One set of sections,representing different levels of the hippocampus, will be used for thevarious stains. The other hemi-brain will be dissected into hippocampus,cortex, thalamus, brain stem, and cerebellum. In this manner, theability of test compounds to reduce neurodegeneration inkainate-injected mice can be assayed.

Example 7 Bioluminescence In Vivo Imaging in Transgenic Reporter Mice

Bioluminescence has been used recently to monitor and quantify geneactivity repeatedly in the same animal in vivo and to study diseaseprogression in peripheral organs with great success.^(46,47) Although,this imaging modality lacks high resolution and cannot be used atpresent to localize signals at the cellular level, it is quantitativeand can faithfully report gene activation if appropriate fusion geneconstructs are used. While initial studies demonstrated the use of thistechnology for the tracking of luciferase expressing bacteria or tumorcells in vivo,^(46,47) more recently transgenic mice were generated thatexpress the Firefly luciferase reporter gene under control the HIV-1LTR⁴⁸, c-fos⁴⁹, or β-lactoglobulin,⁵⁰ or NF-kB promoters/enhancers.

Example 8 Transgenic APP-T41 and Prp-tau Mice

Transgenic mice that overproduce FAD-mutant human APP reproduceimportant aspects of AD, including amyloid plaques, neurodegeneration,and cognitive deficits. APP-T41 mice which overexpressAPP751^(V717I, K670M/N671L) in neurons develop amyloid pathology,neurodegeneration, and cognitive deficits.^(43,44) Mice overexpressinghuman tau protein associated with familial forms of fronto-temporaldementia (a dementia characterized by extensive tangle formation)develop neurofibrillary tangles similar to the ones observed in AD andsuffer from locomotor deficits around 10 months of age.⁴⁵ APP-T41 andPrp-tau^(P301L) mice⁴⁵ can be used to determine the in vivo efficacy oftest compounds in treating AD-like diseases.

APP-T41 mice have low but detectable levels of Aβ in brain and plasma at2-months of age, consistently show Aβ deposits at 5-months of age, andexhibit a prominent pathology at 12-months of age.^(43,44) Testcompounds will be administered to animals at different stages of diseaseprogression to determine when the compound should be administered formaximum effect, how late in disease progression compounds can bedelivered, and the degree of protection afforded by the compounds.Compounds can also be tested for their ability to reverse cognitivedeficits.

Prp-tau^(P301L) mice show consistent tau pathology around 6-months ofage and develop motor deficits around 9-months of age. Motor functioncan be tested using a rotarod and cognitive function can be tested usinga fear conditioning paradigm.

Both short-term and long term studies can be performed using APP-T41 andPrp-tau^(P301L) mice.

Example 9 Behavioral Analysis

Morris Water maze: Mice are trained on a Morris water maze asdescribed⁸⁵. Latency, path length, and proximity scores serve asmeasures of learning. A probe trial will be administered 1 and 7 daysafter training, followed by reversal trials to determine whether theobserved results are due to behavioral inflexibility. Swim speeds willalso be compared.

FIG. 8A shows data obtained in an exemplary Morris water maze experimentcarried out in T41-APP mice. Half males and half females were tested intwo stages: First, in visual platform training, mice swam to a platformmarked with a black and white pole to train the mice to swim to theplatform where they were subsequently rescued by the experimenter. Eachmouse swam 4-times a day for 3 days. Second, in hidden platformtraining, 3-D visual cues were added to the walls of the facility, andthe black and white pole removed. The mice were placed into the tank andswam around the tank to find the hidden platform (up to 90 seconds),using the cues to triangulate their position. Mice were left on theplatform for 10 seconds at the end of the trial to remember the positionof the platform (acquisition phase). Each mouse swam 4 times a day for 6days. Significant differences in acquisition were detected at days 5, 6and 7 using Student's t test and overall differences were significantwith repeated measures ANOVA (p<0.006).

Contextual fear conditioning: To assess cognitive function in r™4510 taumice a Pavlovian fear-conditioning paradigm can be used in addition tothe water maze.⁸⁶⁻⁸⁸ Briefly, after receiving a foot shock coupled withan acoustic stimulus in a brightly lit chamber, mice are exposed to thesame chamber (contextual memory assessment) or placed in a differentlyshaped, scented, and lit box (cued memory assessment), e.g., 24 hours or10 days later. The freezing response of the mice is then quantified.This type of memory test is reliable for assessing deficits incontextual memory and discreet cued memory in mice.^(86,87) It involvesa rapidly acquired form of learning, thought to be a model of humanexplicit memory that appears to involve the hippocampus and that isimpaired in AD.⁸⁹ An additional advantage of fear conditioning overother spatial memory tasks, such as the Morris water maze, is that itminimally relies on motor skills (i.e. stamina and speed), or vision,and allows cognitive testing of mice with slight motor deficits, as isthe case for some transgenic mice.

Example 10 Luciferase Activity in SBE-luc Mouse Hippocampus

SBE-luc mice (T9-7F), 24 months of age, 4-5 mice per group, were giveneither the 11H compound at 30 mg/kg via i.p. injections, or PBS as acontrol. The 11H compound was completely dissolved in PBS at theconcentration of 3 mg/ml. This working solution was given to mice at 10μl/g body weight to achieve the desired level of 30 mg/kg. Five hoursfollowing drug or PBS injection, mice were anesthetized with chloralhydrate and perfused with saline. Brains were dissected and snap-frozen.Hippocampal tissues were homogenized in 100 μl of 1× luciferase assaybuffer and 20 μl of the resulting supernatant was used for luciferaseassays.

Hippocampi from mice injected with the 11H compound demonstrated twicethe levels of activation of the TGF-β reporter gene compared to thoseinjected with PBS (FIG. 1J). These data indicated that the 11H compoundspecifically activated TGF-β reporter gene expression, and therefore,TGF-β signaling, in vivo.

1. A compound having the structure:

wherein R is selected from the group consisting of H, p-CF₃, p-F,p-N(CH₃)₂, and o-OCH₃; R₁ is selected from the group consisting of

—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂N(CH₃)₂, and —(CH₂)_(2 or 3)OCH₃; and R₂ isselected from the group consisting of

and salts thereof, where

in the foregoing structures indicates the point of attachment.
 2. Thecompound of claim 1, wherein when R is equal to H, R₁ is selected fromthe group consisting of

—CH₂CH(CH₃)₂, —CH₂N(CH₃)₂, and —(CH₂)₂OCH₃, and R₂ is selected from thegroup consisting of


3. The compound of claim 1, wherein when R is p-CF₃, R₁ is selected from

—CH(CH₃)₂, —CH₂CH(CH₃)₂, and —(CH₂)_(2 or 3)OCH₃, and R₂ is selectedfrom the group consisting of


4. The compound of claim 1, wherein when R is p-F, R₁ is—(CH₂)_(2 or 3)OCH₃ and R₂ is selected from —CHCH₃Cl,


5. The compound of claim 1, wherein when R is p-N(CH₃)₂, R₁ is selectedfrom —CH(CH₃)₂, —CH₂CH(CH₃)₂, and —(CH₂)₂OCH₃, and R₂ is selected from


6. The compound of claim 1, wherein when R is o-OCH₃, R₁ is either

or —(CH₂)₂OCH₃, and R₂ is either


7. The compound of claim 1, wherein R is hydrogen, R₁ is —CH₂CH(CH₃)₂,and R₂ is


8. The compound of claim 1, wherein R₁ is —(CH₂)₂—OCH₃, R is hydrogen orp-CF₃, and R₂ is either


9. The compound of claim 1 corresponding to the structure,

wherein the tryptamine is further substituted at one or more of the 4,5, 6, or 7 positions, and wherein the substitution is independentlyselected, for each position, from the group consisting of fluoro,methyl, hydroxyl, methoxy, and benzyloxy.
 10. The compound of claim 1corresponding to the structure,

wherein the tryptamine is further substituted at one or more of the α or1 positions, and wherein the substitution is independently selected, foreach position, from the group consisting of fluoro, methyl, hydroxyl,methoxy, and benzyloxy.
 11. The compound of claim 1 having thestructure:


12. A pharmaceutical composition comprising a compound of claim 1 and anadditive selected from the group consisting of pharmaceuticallyacceptable carriers, binders, disintegrating agents, surfactants,antioxidants, and lubricants.
 13. The pharmaceutical composition ofclaim 12, in the form of a tablet, capsule, or a liquid for oraladministration.